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Cloning,expression, and characterization of catechol 1,2-dioxygenase from a phenol-degrading Candida tropicalis JH8 strain
Authors:Yan Long  Sheng Yang  Li Cheng
Institution:1. College of Life Sciences, Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), State Key Laboratory of Virology, Wuhan University, Wuhan, China;2. Hubei Provincial Cooperative Innovation Center of Industrial Fermentation, Wuhan University, Wuhan, China;3. College of Life Sciences, Hubei University, Wuhan, China
Abstract:The sequence cato encoding catechol 1,2-dioxygenase from Candida tropicalis JH8 was cloned, sequenced, and expressed in Escherichia coli. The sequence cato contained an ORF of 858?bp encoding a polypeptide of 285?amino acid residues. The recombinant catechol 1,2-dioxygenase exists as a homodimer structure with a subunit molecular mass of 32 KD. Recombinant catechol 1,2-dioxygenase was unstable below pH 5.0 and stable from pH 7.0 to 9.0; its optimum pH was at 7.5. The optimum temperature for the enzyme was 30°C, and it possessed a thermophilic activity within a broad temperature range. Under the optimal conditions with catechol as substrate, the Km and Vmax of recombinant catechol 1,2-dioxygenase were 9.2?µM and 0.987?µM/min, respectively. This is the first article presenting cloning and expressing in E. coli of catechol 1,2-dioxygenase from C. tropicalis and characterization of the recombinant catechol 1,2-dioxygenase.
Keywords:Candida tropicalis JH8  catechol 1  2-dioxygenase  cloning and expression  enzymatic properties
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